The current study assesses the water quality and ecological health of the Bhargirathi River, the source tributary of the Ganga River, which is serially fragmented by four hydroelectric projects (HEPs), using benthic macroinvertebrate community as bioindicators. Six stations were selected for physicochemical parameters and benthic macroinvertebrate sample collection: S1 an upstream station in the natural flow condition; S2, S4 and S5 selected at varying distances downstream (d/s) the diversion or barrage receiving constant low flows; and S3 and S6 situated d/s the powerhouses receiving the regulated discharge. The Biological Monitoring Working Party (BMWP) score was used to assess the level of organic pollution; the functional feeding group (FFG) analysis to evaluate the functional state; and the Lotic Invertebrate Flow Evaluation Index (LIFE index) to assess the flow alterations. The water temperature (°C) showed a significant (p < 0.05) rise at the S2, S4 and S5, dissolved oxygen and water velocity revealed significant decline in the modified sections. BMWP scores (41–70, November to April) reflect water quality class III at the reference station S1. In contrast, the water quality class significantly declined to class IV at S2, S3 and S4 and class V at S6 indicating the enhanced organic pollution in these stations. The water quality improved (Class III at S5, as the river traverses away from the diversion. FFG analysis reveals rise in the share of filter collectors at S2, S4 and S5, the flow deficient sections indicating heterotrophic state of the river in contrast to relatively autotrophic at S1. The LIFE index, indicated the colonization of the taxa preference for low flows at the S2, S4 and S5 and fast to rapid flows at S3 and S5 reflecting the alterations. The findings suggest the severe impacts on the HEP modified riverine sections deteriorating the ecosystem health suggesting regular monitoring of these sections and allocation of the required environmental flow to sustain ecosystem structure. Furthermore, the study suggests ministry of Jal shakti Govt. of India to incorporate the hydropower-impacted section for the identification of polluted sections of the river for management and conservation of riverine ecosystem.